海马放射状胶质细胞的体外诱导激活及向胆碱能神经元的分化
发布时间：2019-05-24 17:27
【摘要】：第一部分：海马放射状胶质细胞的体外诱导激活及其神经干细胞样特性 目的探讨海马放射状胶质细胞在切割穹窿海马伞去神经支配海马提取液诱导下的形态学变化以及细胞增殖、胚胎源性和向神经元及胶质细胞分化的神经干细胞样特性。 方法取生后1 d的SD大鼠海马组织,应用差速贴壁及摇床振荡法分离纯化海马放射状胶质细胞。将纯化的海马放射状胶质细胞接种于24孔培养板中,分成切割组和正常组,切割组加入含5%(v/v)切割穹窿海马伞去神经支配海马提取液的DMEM/F12培养液,正常组加入含5%(v/v)正常侧海马提取液的DMEM/F12培养液,同时在两组中加入5-溴-2-脱氧尿嘧啶(BrdU)标记增殖的细胞,并分别于培养后1、3、7和14 d时行BLBP免疫荧光检测和Hoechst标记；同时用BLBP/BrdU、BLBP/nestin、BLBP/MAP-2、BLBP/GFAP、BLBP/CNP免疫荧光双标技术观察其增殖特性和胚胎源性以及向神经元、星型胶质细胞、少突胶质细胞分化的情况,检测BrdU阳性细胞占BLBP阳性细胞的百分比、BLBP阳性细胞的周长和面积,以及两组中BLBP/nestin、BLBP/MAP-2、BLBP/GFAP、BLBP/CNP双标细胞占BLBP阳性细胞的百分比。应用Stata10.0统计软件行组间比较。 结果BLBP免疫荧光检测结果显示,通过上述的纯化培养方法,我们获得了几近100%的海马放射状胶质细胞,BLBP在细胞浆、细胞核及突起中均有表达。切割组BrdU阳性细胞占BLBP阳性细胞的百分比明显高于正常组(切割组：56.86±8.52%；正常组：31.11±4.28%,P0.01)。接种1 d后,两组细胞的胞体均较小,突起较短较细；3 d时,与正常组相比,切割组BLBP阳性细胞的胞体稍变大,突起稍变得粗而长；7 d时,与正常组相比,切割组BLBP阳性细胞的胞体明显变大,突起明显变粗、变长且交织成网状；14 d时两组细胞的胞体和突起均开始变细变短,正常组较为明显。两组各d BLBP阳性细胞的周长和面积的统计分析结果表明除1 d时两组BLBP阳性细胞的周长和面积无明显差异(P0.05)外,其余各d切割组BLBP阳性细胞的周长和面积均高于正常组(P0.01)。切割组nestin阳性细胞占BLBP阳性细胞的百分比也明显高于正常组(切割组：57.92±17.93%；正常组：23.26±9.85%,P0.01)。并可见切割组中较多的BLBP阳性细胞向MAP-2阳性的神经元分化(切割组：46.13±14.92%；正常组：29.13±10.07%,P0.05),虽然两组向GFAP阳性的星型胶质细胞和CNP阳性的少突胶质细胞分化在数量上无明显差异,但切割组中两种胶质细胞的胞体明显增大、突起明显丰富。 结论切割穹窿海马伞去神经支配海马提取液不仅可以明显诱导BLBP阳性放射状胶质细胞增殖、胞体明显增大、突起变粗变长呈“激活”状态,具胚胎源性,而且还可使其向神经元、星型胶质细胞和少突胶质细胞分化,表现为神经干细胞样特性。 第二部分：去神经支配海马提取液诱导海马放射状胶质细胞向胆碱能神经元的分化 目的在体外细胞培养中加入切割穹窿海马伞去神经支配海马提取液,模拟在体海马神经再生微环境,观察海马放射状胶质细胞向胆碱能神经元分化的情况。 方法将纯化的海马放射状胶质细胞分成切割组和正常组,切割组加入含5%(v/v)切割穹窿海马伞去神经支配海马提取液的DMEM/F12培养液,正常组加入含5%(v/v)正常侧海马提取液的DMEM/F12培养液,分别在培养7d后应用BLBP/ChAT免疫荧光双标、Real-time PCR及Western blot等技术观察两组放射状胶质细胞向胆碱能神经元的分化,以及表达ChAT mRNA和蛋白的情况。 结果ChAT免疫荧光结果显示,加入切割穹窿海马伞侧海马提取液后,与正常组相比,切割组中ChAT阳性的胆碱能神经元占BLBP阳性细胞的比例(41.62±9.97%)明显多于正常组(16.08±7.31%) (P0.01),且切割组中ChAT阳性的胆碱能神经元的胞体较正常组明显增大,突起明显增粗增长。切割组ChAT mRNA水平明显高于正常组,约是正常组的5倍(P0.01)。ChAT蛋白的表达量虽较低,但切割组(0.1141±0.0380)仍高于正常组(0.0423±0.0106)(P0.05)。 结论切割穹窿海马伞侧海马提取液可明显诱导BLBP阳性放射状胶质细胞向胆碱能神经元分化。
[Abstract]:The first part: the in vitro induction activation of the radial glial cells in the hippocampus and its neural stem cell-like characteristics Objective To study the morphological changes and the increase of the cells in the hippocampus of the hippocampus of the hippocampus from the hippocampus of the hippocampus. Neural stem cell samples for the differentiation of colonizing, embryonic-derived and neuron-and glial cells Sex. The rat hippocampal tissue was isolated and purified by differential malapposition and shaking method in the hippocampus of SD rats. culturing the purified hippocampal radial glial cells in a 24-well culture plate, dividing into a cutting group and a normal group, adding a DMEM/ F12 culture solution containing 5% (v/ v) cutting a hole-hole hippocampal umbrella to the neurodominant hippocampus extract, and adding DMEM/ F12 containing 5% (v/ v) normal-side hippocampal extract in the normal group; The cells were labeled with 5-bromo-2-deoxy-1-detrusor (BrdU) in both groups. The BLBP/ BrdU, BLBP/ nestin, BLBP/ MAP-2, BLBP/ G were also used. The proliferation and embryo-origin of the BLBP-positive cells, the percentage of BLBP-positive cells, the perimeter and the area of the BLBP-positive cells and the BLBP/ nestin, BLBP/ MAP-2 and BLBP/ G in the two groups were examined by means of the two-standard technique, such as FAP, BLBP/ CNP. FAP, BLBP/ CNP double-labeled cells account for BLBP positive cells %. Applying the Staa10.0 Statistics Software Line Group The results of the results of the BLBP immunofluorescence test show that, by the above-mentioned purification and culture method, we obtained nearly 100% of the hippocampal radial glial cells, and the BLBP is in the cytoplasm, nucleus and protrusion of the cells. The percentage of BrdU positive cells in the cut group was significantly higher than that in the normal group (56.86-8.52%, normal group: 31.11-4.28%, P 0.01) After 1 day of inoculation, the cell bodies of the two groups were small and the protrusions were shorter and thinner; when compared with the normal group, the cells of the BLBP positive cells of the cut group were slightly larger and the protrusions were slightly thicker and longer; at the time of 7 d, the cells of the BLBP positive cells of the cut group were compared with the normal group. The body of the two groups of cells began to become thinner and shorter, and the body and the protrusion of the two groups of cells started to become shorter and normal at the time of 14 d. The results of the statistical analysis of the perimeter and area of each d BLBP positive cells in the two groups showed no significant difference in the circumference and the area of the BLBP positive cells in the two groups (P <0.05), and the perimeter and area of the BLBP positive cells in the remaining d groups were higher than that in the normal group (P The percentage of nestin positive cells in the cut group was significantly higher than that in the normal group (group: 57.92-17.93%, normal group: 23.26-9.85%, P 0.01). It was found that more of the BLBP positive cells in the cut group were differentiated into the MAP-2-positive neurons (group: 46.13-14.92%, normal group: 29.13-10.07%, P0.05). There was no significant difference, but the cell bodies of the two kinds of glial cells in the cutting group were obviously increased, and the process of the process Conclusion The removal of the hippocampal extract from the hippocampus of the rat hippocampus can not only obviously induce the proliferation of the BLBP-positive radial glial cells, the cell body is obviously increased, the growth of the protrusion becomes the "activate" state, the embryo-derived, but also can differentiate into neurons, astrocytes, and oligodendrocytes and act as a god Stem cell-like properties. Part 2: The removal of the hippocampal extract from the hippocampus in the innervation of the hippocampal extract. The purpose of the differentiation of cholinergic neurons in the in vitro cell culture is to add a cut hole in the hippocampus of the hippocampus to innervate the hippocampal extract, to simulate In that microenvironment of the neural regeneration in the hippocampus of the body, the radial glial cells of the hippocampus were observe. The method of the invention comprises the following steps of: dividing the purified hippocampal radial glial cells into a cutting group and a normal group, adding a DMEM/ F12 culture solution containing 5% (v/ v) cutting a hole-hole in the hippocampus of the hippocampus, and adding 5% (v/ v) normal-side hippocampal extraction to the normal group; The differentiation of the two groups of radial glial cells to the cholinergic neurons was observed by BLBP/ ChAT immunofluorescence double-label, Real-time PCR and Western blot. Results The results showed that the proportion of ChAT-positive cholinergic neurons in the cut group (41.62% 9.97%) was significantly higher than that of the normal group (16.0%) compared with the normal group (16.0%). 8 (7.31%) (P0.01), and the cells of ChAT-positive cholinergic neurons in the cut group were positive The expression of ChAT mRNA in the cutting group was significantly higher than that in the normal group (P0.01). The expression of ChAT protein was lower, but the cleavage group (0.1141-0.0380) was still higher than that in the normal group (0.0423. Conclusion: It is concluded that the extract of the hippocampus on the side of the hippocampus can induce BLBP-yang obviously.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2485044
[Abstract]:The first part: the in vitro induction activation of the radial glial cells in the hippocampus and its neural stem cell-like characteristics Objective To study the morphological changes and the increase of the cells in the hippocampus of the hippocampus of the hippocampus from the hippocampus of the hippocampus. Neural stem cell samples for the differentiation of colonizing, embryonic-derived and neuron-and glial cells Sex. The rat hippocampal tissue was isolated and purified by differential malapposition and shaking method in the hippocampus of SD rats. culturing the purified hippocampal radial glial cells in a 24-well culture plate, dividing into a cutting group and a normal group, adding a DMEM/ F12 culture solution containing 5% (v/ v) cutting a hole-hole hippocampal umbrella to the neurodominant hippocampus extract, and adding DMEM/ F12 containing 5% (v/ v) normal-side hippocampal extract in the normal group; The cells were labeled with 5-bromo-2-deoxy-1-detrusor (BrdU) in both groups. The BLBP/ BrdU, BLBP/ nestin, BLBP/ MAP-2, BLBP/ G were also used. The proliferation and embryo-origin of the BLBP-positive cells, the percentage of BLBP-positive cells, the perimeter and the area of the BLBP-positive cells and the BLBP/ nestin, BLBP/ MAP-2 and BLBP/ G in the two groups were examined by means of the two-standard technique, such as FAP, BLBP/ CNP. FAP, BLBP/ CNP double-labeled cells account for BLBP positive cells %. Applying the Staa10.0 Statistics Software Line Group The results of the results of the BLBP immunofluorescence test show that, by the above-mentioned purification and culture method, we obtained nearly 100% of the hippocampal radial glial cells, and the BLBP is in the cytoplasm, nucleus and protrusion of the cells. The percentage of BrdU positive cells in the cut group was significantly higher than that in the normal group (56.86-8.52%, normal group: 31.11-4.28%, P 0.01) After 1 day of inoculation, the cell bodies of the two groups were small and the protrusions were shorter and thinner; when compared with the normal group, the cells of the BLBP positive cells of the cut group were slightly larger and the protrusions were slightly thicker and longer; at the time of 7 d, the cells of the BLBP positive cells of the cut group were compared with the normal group. The body of the two groups of cells began to become thinner and shorter, and the body and the protrusion of the two groups of cells started to become shorter and normal at the time of 14 d. The results of the statistical analysis of the perimeter and area of each d BLBP positive cells in the two groups showed no significant difference in the circumference and the area of the BLBP positive cells in the two groups (P <0.05), and the perimeter and area of the BLBP positive cells in the remaining d groups were higher than that in the normal group (P The percentage of nestin positive cells in the cut group was significantly higher than that in the normal group (group: 57.92-17.93%, normal group: 23.26-9.85%, P 0.01). It was found that more of the BLBP positive cells in the cut group were differentiated into the MAP-2-positive neurons (group: 46.13-14.92%, normal group: 29.13-10.07%, P0.05). There was no significant difference, but the cell bodies of the two kinds of glial cells in the cutting group were obviously increased, and the process of the process Conclusion The removal of the hippocampal extract from the hippocampus of the rat hippocampus can not only obviously induce the proliferation of the BLBP-positive radial glial cells, the cell body is obviously increased, the growth of the protrusion becomes the "activate" state, the embryo-derived, but also can differentiate into neurons, astrocytes, and oligodendrocytes and act as a god Stem cell-like properties. Part 2: The removal of the hippocampal extract from the hippocampus in the innervation of the hippocampal extract. The purpose of the differentiation of cholinergic neurons in the in vitro cell culture is to add a cut hole in the hippocampus of the hippocampus to innervate the hippocampal extract, to simulate In that microenvironment of the neural regeneration in the hippocampus of the body, the radial glial cells of the hippocampus were observe. The method of the invention comprises the following steps of: dividing the purified hippocampal radial glial cells into a cutting group and a normal group, adding a DMEM/ F12 culture solution containing 5% (v/ v) cutting a hole-hole in the hippocampus of the hippocampus, and adding 5% (v/ v) normal-side hippocampal extraction to the normal group; The differentiation of the two groups of radial glial cells to the cholinergic neurons was observed by BLBP/ ChAT immunofluorescence double-label, Real-time PCR and Western blot. Results The results showed that the proportion of ChAT-positive cholinergic neurons in the cut group (41.62% 9.97%) was significantly higher than that of the normal group (16.0%) compared with the normal group (16.0%). 8 (7.31%) (P0.01), and the cells of ChAT-positive cholinergic neurons in the cut group were positive The expression of ChAT mRNA in the cutting group was significantly higher than that in the normal group (P0.01). The expression of ChAT protein was lower, but the cleavage group (0.1141-0.0380) was still higher than that in the normal group (0.0423. Conclusion: It is concluded that the extract of the hippocampus on the side of the hippocampus can induce BLBP-yang obviously.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前10条
1 金国华,张新化,田美玲,秦建兵,黄镇,徐慧君;穹窿海马伞切割侧海马提取液对神经干细胞分化为神经元的促进作用[J];解剖学报;2004年02期
2 朱蕙霞,陈蓉,金国华,秦建兵,田美玲;非变性聚丙烯酰胺凝胶电泳在大鼠海马组织蛋白分离中的应用[J];南通医学院学报;2003年04期
3 朱蕙霞;秦建兵;田美玲;陈蓉;金国华;;切割海马伞海马中56kD差异蛋白的质谱分析[J];南通大学学报(医学版);2006年06期
4 赵善廷,邓锦波;大鼠海马结构的组成、细胞类型及神经纤维联系 Ⅰ.齿状回[J];神经解剖学杂志;1999年01期
5 龙大宏,杨丹迪,李佳楣,许孟杰,汪华侨;穹窿海马伞损伤对大鼠学习记忆和海马GFAP阳性神经胶质细胞的影响[J];神经解剖学杂志;2002年01期
6 金国华,张新化,田美玲,黄镇,秦建兵,徐慧君;大鼠海马内移植神经干细胞的存活和迁移[J];神经解剖学杂志;2003年04期
7 张新化,金国华,秦建兵,田美玲,黄镇,徐慧君;穹窿海马伞切割侧海马对植入神经干细胞分化为神经元的影响[J];神经解剖学杂志;2004年04期
8 陈蓉,金国华,田美玲,朱蕙霞,秦建兵,谭雪锋,徐慧君;海马中56kD蛋白诱导人神经干细胞迁移的作用[J];神经解剖学杂志;2005年04期
9 金国华;陈蓉;田美玲;秦建兵;谭雪锋;朱蕙霞;徐慧君;;海马中56 kD蛋白诱导人神经干细胞向神经元分化的作用[J];神经解剖学杂志;2006年04期
10 徐璐璐;王磊;衣昕;秦建兵;田美玲;金国华;;切割穹窿海马伞大鼠海马内BLBP的表达变化[J];神经解剖学杂志;2008年06期
，本文编号：2485044
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2485044.html
最近更新
教材专著
